Molecular Biology provides powerful tools for engineering and recombination of nucleic acids. Restriction enzymes, site-directed mutagenesis, various polymerase-chain-reaction (PCR)-based strategies, synthesis-based strategies, homologous recombination, and other approaches, are all employed in the production of engineered nucleic acids and/or the variation of nucleic acid sequences. New techniques, or improved versions of existing techniques, continue to be developed. However, further room for improvement exists.
A variety of techniques have been developed for generating diversity in or among nucleic acid sequences. Some such techniques involve recombination between or among related nucleic acid sequences, typically followed by selection of desired recombined sequences (for example, see Patten et al., U.S. Pat. Nos. 6,579,678 and 6,613,514). Such approaches have significant drawbacks, however, not the least of which is that due to the stochastic nature of recombination, the practitioner must rely on a chance recombination event to generate a particular nucleic acid sequence. Furthermore, one or more of the parental molecules may fail to undergo recombination or may be reconstituted in a recombination reaction, such that extensive screening is required to identify new recombinants of interest.
Hence, there is still a need for improved methods of generating engineered nucleic acid sequences and of generating diversity in a population of nucleic acid molecules.